Babies born weighing less than 2,500 g are considered low birth weight (LBW), and are at increased risk for serious health problems as neonates, lasting disabilities and even death. Certain LBW babies can be further classified into Very Low Birth Weight (VLBW) babies, born at less than 1,500 g, and Extremely Low Birth Weight (ELBW) babies, born at less than 1000 g. The rate of LBW neonates shows differences around the world. For example, the World Health Organization (WHO) estimated that 16.5% of births in less developed regions in the year 2000 were LBW. In contrast, around 1 of every 12 (8.3%) babies born in 2005 in the United States was born LBW (Martin et al, 2007; National Vital Statistics Reports, 56 (6)) and in England and Wales the overall rate of LBW babies has been reported as 7.3% (Doyle, 2000; BMJ, 320: 941-942). The rate of LBW babies is increasing, particularly in more developed regions such as the United States, believed to result predominantly from an increase in preterm delivery of artificially conceived multiple pregnancies.
Many LBW babies require specialized care in Newborn Intensive Care Units (NICUs) as they are especially susceptible to health problems (for example, as reported in McIntire et al, 1999; N Engl J Med, 340: 1234-1238) including respiratory distress syndrome (RDS), cyanotic attacks, bleeding in the brain (intraventricular hemorrhage, IVH), cerebral palsy, heart problems such as patent ductus artiousus (PDA), hypocalcemia, hypoglycaemia, intestinal problems such as necrotizing enterocolitis (NEC), jaundice and retinal-development problems such as retinopathy of prematurity (ROP). A number of studies have found that the cost of care for neonatal care rises steeply with decreasing birth weight, with a US study estimating neonatal costs to be $224,400 for a newborn with a birth-weight of 500-700 g, compared to only $1000 for a baby born with a birth-weight over 3,000 g, and one estimate of over $50 billion in annual costs for such care provided in the United States. Beyond such acute care, being born underweight has been reported to be associated with a number of mid-term health problems, including: (a) poor weight gain and head growth in infancy (Gutbrod et al, 2000; Arch Dis Child Fetal Neonatal Ed, 82: 208-214); (b) developmental delay and later language problems in early childhood (Marlow et al, 2005; N Engl J Med, 352: 9-19); (c) neurological abnormalities; and (d) increased incidence of deafness. Some studies also suggest that individuals born LBW may be at increased risk for certain chronic conditions in adulthood, including high blood pressure, type-2 diabetes and heart disease.
There are two main reasons why a baby may be born with low birth weight: (1) premature birth—a normal pregnancy lasts for about 40 weeks (38-42 weeks), and the WHO defines prematurity as a baby born before 37 full-weeks from the first day of the last menstrual period. The earlier a baby is born, the less it is likely to weigh; and (2) fetal growth restriction—babies that may be full-term but are underweight, also known as small-for-gestational age (SGA) or small-for-date babies. Some of these babies are small simply because their parents are small (and these babies are often healthy), while others have low birth weight because something has slowed or halted their growth in the uterus (or intra-uterine growth retardation, IUGR). Some babies are both premature and have suffered IUGR, and these babies are particularly at high risk for health problems such as those described above.
Recently, the WHO has systematically reviewed the worldwide incidence of preterm birth (Beck et al, 2010; Bull World Health Organ, 88: 31-38), and they estimate that in 2005 12.9 million births, or 9.6% of all births worldwide, were preterm. Approximately 11 million (85%) of these preterm births were concentrated to Africa and Asia, while about 0.5 million occurred in each of Europe and North America (excluding Mexico) and 0.9 million in Latin America and the Caribbean. The highest rates of preterm birth were in Africa and North America (11.9% and 10.6% of all births, respectively), and the lowest were in Europe (6.2%). The relatively high rate of preterm births estimated for North America equates to an estimated absolute number of 480,000 preterm births in 2005, and despite the relatively lower rate, still equates to an estimated 466,000 preterm births in Europe during the same year. Preterm birth rates available from some developed countries, such as the United Kingdom, the United States and the Scandinavian countries, show a dramatic rise over the past 20 years (e.g. Callaghan et al, 2006; Pediatrics, 118: 1566-1573). Factors possibly contributing to but not completely explaining this upward trend include increasing rates of multiple births, greater use of assisted re-production techniques, increases in the proportion of births among women over 34 years of age and changes in clinical practices, such as more frequent use of elective Caesarean section.
Preterm babies are generally susceptible to the same health problems as LBW babies, with the severity of the problems increasing with degree of prematurity: a baby born at 36 weeks will probably be a little slow to feed; a baby born before 33 weeks will have more serious problems including, possibly, immature lungs; while a birth before 28 weeks causes very significant problems but the survival rate is quite remarkable. Data suggest 90% survival if born over 800 g, 50% survival if over 500 g and 80% survival if born before 28 weeks; although these figures may also hide significant disability in survivors. For example, severe problems such as cerebral palsy, blindness and deafness may affect as many as 10 to 15% of significantly premature babies, about 1 in 4 babies with birth weight below 1.5 kg has peripheral or central hearing impairment or both (Jiang et al, 2001; Acta Paediatr, 90 1411-1415) and 66% of babies under 1.25 kg develop ROP (Allin et al, 2006; Pediatrics, 117: 309-316).
Pancreas and liver functions are not fully developed at birth, and in premature infants this is particularly notable. Lindquist and Hernell (1990; Curr Opin Clin Nutr Metab Care, 13: 314-320) have recently reviewed the subject of lipid digestion and absorption in early life. Breast-fed infants digest and absorb fat (and importantly long-chain polyunsaturated fatty acids, LCPUFAs) more efficiently than formula-fed infants (Bernback et al, 1990; J Clin Invest, 85:1221-1226; Carnielli et al, 1998; Am J Clin Nutr, 67: 97-103). In addition to infant formulas of similar fat composition, mother's milk also contains a broad-specificity lipase, bile-salt stimulate lipase (BSSL) (EC 3.1.1.13) that promotes highly efficient fat absorption from human milk.
BSSL is a naturally occurring pancreatic enzyme which is activated by bile salts in the duodenum and participates in the hydrolysis of lipids together with other lipases. In early infancy, and especially in the preterm infant, pancreatic exocrine functions are not fully developed (Manson & Weaver, 1997; Arch Dis Child Fetal Neonatal Ed, 76: 206-211). Hence, in the preterm pancreas, expression of pancreatic lipases is low compared to adult pancreas (Lombardo, 2001; Biochim Biophys Acta, 1533: 1-28; Li et al 1007; Pediatr Res, 62: 537-541). Therefore, the BSSL present in breast milk is an important lipase for these infants; the low level of pancreatic lipase is compensated for by expression of BSSL in the lactating mammary gland and secretion of the enzyme with the mother's milk. The human lactating mammary gland synthesizes and secretes BSSL that, after specific activation by primary bile salts in the lower intestine of the baby, contributes to the breast-fed infant's endogenous capacity for intestinal fat digestion.
BSSL is believed to have a broader substrate specificity than most lipases. Not only is the enzyme capable of completely hydrolyzing all three fatty acids of triglycerides (triacylglycerols, TGs), but also fat soluble vitamin esters such as vitamin A as well as cholesteryl esters. Thus, BSSL drives the intraluminal lipolysis toward completion and results in the formation of glycerol and free fatty acids (FFAs), including long-chain polyunsaturated fatty acids (LCPUFAs), the latter being indispensable building blocks for the developing central nervous system (Hernell, 1975; Eur J Clin Invest, 5: 267-272; Bernback et al, 1990; Hernell et al, 1993; J Pediat Gastro Nutr, 16: 426-431; Chen et al, 1994; Biochem Biophys Acta, 1210: 239-243). BSSL shows optimal activity at a pH of 8-8.5 and is more stable in acid environments than pancreatic lipase. BSSL is resistant to degradation by pepsin at physiological concentrations. BSSL accounts for about 1% of the total protein in milk and is present at concentrations from 0.1-0.2 g/L (Blackberg et al, 1987; FEBS Lett, 217: 37-41; Wang & Johnson, 1983; Anal Biochem, 133: 457-461; Stromqvist et al, 1997; Arch Biochem Biophys, 347: 30-36). The levels of BSSL in human milk are similar throughout the day (Freed et al, 1986; J Pediatr Gastroenterol Nutr, 5: 938-942) and BSSL production in human milk is maintained for at least 3 months (Hernell et al, 1977; Am J Clin Nutr, 30: 508-511) although concentrations of BSSL may decline with duration of lactation (Torres et al, 2001; J Natl Med Assoc, 93: 201-207). Triglycerides comprise about 98% or more of all lipids in human milk or formula and thereby account for about 50% of the energy content.
The superiority of human milk as a nutritional source for term as well as preterm infants has been manifested in many studies and expert group recommendations. Accordingly, the recommended feeding method world-wide is breastfeeding. Neither is however, breastfeeding nor feeding the mother's own breast milk always possible or recommended for medical reasons—and breastfeeding may not be practiced for a number of other reasons—in each case as discussed elsewhere herein.
Despite dropping from about 60% to about 50% in the 1980s (Foss & Southwell, 2006; Int Breastfeeding J 1: 10), the percentage of US women initiating breastfeeding increased from the 1990s and was reported to be about 74% in 2004 (Scanlon et al, 2007, in CDC Morbidity and Mortality Weekly Report, 2 Aug. 2007). However, the percentage of women continuing breast feeding appears to drop substantially after initiation in the early postpartum period, with this study reporting that in 2004 only 42% and 21% of women were still breastfeeding after 6 and 12 months, respectively. Data from Sheffield in the UK showed the same trends, with a drop from about 70% to 50% during the 1980s but that only 35% to 30% (during the same period) of such women were actually doing so at one month after birth (Emery et al, 1990, Arch Dis Childhd, 65: 369-372). The percentage of women who exclusively breastfeed is even lower, and overall for infants born in the US in 2004 only about 31% and 11% of women were exclusively breastfeeding through ages 3 and 6 months, respectively, and with significant disparities between subgroups of these women; rates of exclusive breast feeding through age 3 months were lowest among black infants (20%) and among infants of mothers who were aged <20 years (17%), had a high school education or less (23% and 24%, respectively), were unmarried (19%) resided in rural areas (24%) and had an income-to-poverty ratio of <100% (24%) (Scanlon et al, 2007). Indeed, significant national and cultural differences in breastfeeding exist. Emery & coworkers (1990) reported a significantly lower percentage of Asian women than white women intending to breastfeed in Sheffield UK. Furthermore, Singh (2010; Eur J Sci Res, 40: 404-422) has reported that in Brazil, the mean duration of exclusive breastfeeding is only 28.9 days, in Malaysia only 25% or infants are exclusively breast fed at 2 months and in Bogota and Nairobi this percentage is 12% and 21% or infants, respectively.
In cases where the infant is not breast-fed, infant formula or banked and non-banked pasteurized and/or frozen breast milk is often used. All are, however, in some respects nutritionally suboptimal for newborn infants.
Due to risks of viral infection (human immunodeficiency virus [HIV], cytomegalovirus [CMV], hepatitis) and to a lesser degree transmission of pathogenic bacteria, donor milk used in so-called milk banks is generally pasteurized before it is used. However, BSSL is inactivated during pasteurization of human milk (Björksten et al, 1980; Br Med J, 201: 267-272); nor is it present in any of the many different formulas that exist for the nutrition of pre- or full-term neonates. It has been shown that fat absorption, weight gain and linear growth is higher in infants fed fresh compared to pasteurized breast milk (Andersson et al. 2007; Acta Paediatr, 96: 1445-1449; Williams et al, 1978; Arch Dis Child 43: 555-563). This is one reason why it has been advocated that newborn infants, particularly preterm infants, that cannot be fed their own mothers milk should be fed non-pasteurized milk from other mothers (Björksten et al, 1980).
Hamosh (1983; J Ped Gastro Nutr, 2: 248-251) reported that BSSL enzyme activity is present in fresh breast milk of women who delivered at 26 to 30 weeks. This report further described that milk specimens stored at −20 or −10° C. showed a slow loss in BSSL activity, but a more dramatic loss of bile-salt dependency on activity after only three weeks storage at −10° C. which may contribute to hydrolysis of milk lipids even during storage of breast milk at −20° C.
Milk bile-salt-stimulated lipase has been found only in the milk of certain species, namely humans, gorillas, cats and dogs (Freed, et al, 1986; Biochim Biophys Acta, 878: 209-215). Milk bile-salt-stimulated lipase is not produced by cows, horses, rats, rabbits, goats, pigs or Rhesus monkeys (Blackberg et al, 1980; Freudenberg, 1966; Experientia, 22: 317).
Native human milk BSSL (hBSSL-MAM) has been purified to homogeneity, as reported by Blackberg & Hernell (1981; Eur J Biochem, 116: 221-225) and Wang & Johnson (1983), and the cDNA sequence of human BSSL was identified by Nilsson (1990; Eur J Biochem, 192: 543-550) and disclosed in WO 91/15234 and WO 91/18923. Characterization and sequence studies from several laboratories concluded that the proteins hBSSL-MAM and the pancreas carboxylic ester hydrolase (CEH) (also known as pancreatic BSSL) are both products of the same gene (for example, Baba et al, 1991; Biochem, 30: 500-510 Hui et al, 1990; FEBS Lett, 276: 131-134; Reue et al, 1991; J Lipid Res, 32: 267-276).
Following the isolation of the cDNA sequence, recombinant human BSSL (rhBSSL), as well as variants thereof, has been produced including in transgenic sheep (rhBSSL-OVI); such as described in U.S. Pat. No. 5,716,817, WO 94/20610 and WO 99/54443. Production of proteins for therapeutic use using transgenic animals has been met with significant safety, scientific, regulatory and ethical resistance. Indeed, to date there is no approved therapeutic product on the US or EU market that has been produced from transgenic sheep, and only two medical products produced from other transgenic animals have so far been approved: ATRYN (recombinant antithrombin) produced from transgenic goats, and RUCONEST (recombinant component 1 esterase inhibitor) produced from transgenic rabbits. Proteins produced in such a manner (to be expressed in mammary tissue and excreted in milk) can be contaminated with components naturally found in the milk of these animals, such as whey or non-human milk or whey proteins, which may cause safety issues if such proteins are used for human use in certain individuals, such as those intolerant or allergic to milk-based components or products.
It has long been known (at least by the mid-1960s) that the addition of lipase or esterase-containing tissue-extracts to milk-based food is useful in the treatment of scours in animals (CA 662815 & U.S. Pat. No. 3,081,225). Also, U.S. Pat. No. 326,150 suggests the use of exogenous lipases for the treatment of celiac disease or malabsorption syndrome in humans, including in young children. The methods described therein involve the extraction and use of a largely uncharacterized mixture of enzymes from tissues such as the tongue and other oral tissues of calves, kid-goats and lambs.
With reference to infant feeding practice, in particular the feeding of LBW infants, it has long been promoted that fresh human breast milk is the most suitable feed for LBW infants. This is based on studies such as the early work by Williams et al (1978) who showed that heat-treatment of human milk reduced fat absorption by approximately one-third (compared to raw human milk) in an experimental study of seven VLBW preterm infants (less than 1.3 Kg) aged between 3 and 6 weeks, fed for three consecutive weeks with raw, pasteurized and boiled human milk, each for one week. This study made the suggestion that the improvement in fat absorption may be related to the preservation of milk lipases in the raw, compared to the heat-treated, human milk. Of note is that this study described that all infants gained weight most rapidly during the week in which they were fed raw milk; with the mean weight gain (reported in g gained per week per 100 mL milk consumed) during this period approximately one third greater than the similar periods during which pasteurized or boiled milk was administered. In a larger (but shorter) study reported by Alemi (1980; Pediatrics. 68: 484-489), fat excretion was studied in 15 VLBW infants, born with a birth-weight of between 660 and 1,695 g and a gestational age of 26 to 33 weeks, and the study started at 7 to 44 days after birth. Fat excretion was lower in those infants fed a mixture of human milk and formula for 72 hours compared to the infants fed formula only. More recently, Andersson & coworkers (2007) reported in a randomized study that pasteurization of mother's own milk reduced fat absorption and growth in preterm infants, and proposed that these effects were due to inactivation of milk-based BSSL by pasteurization. Of note is that the reported range of coefficient of fat absorption (CFA) from a number of studies, including those above, are wide; both from human milk and from formulas. This can partly be explained by the amount and composition of fat given, and partly by large interindividual differences in the capacity to utilize dietary fat in preterm newborns, but it also reflects a considerable difficulty in correctly assessing CFA (Hernell, 1999; J Pediatr, 136: 407-409).
One animal model study has attempted to investigate the effects on infant growth by the addition on exogenous BSSL to neonatal food (Wang et al, 1989; Am J Clin Nutr, 49: 457-463). This study involved the addition of purified human BSSL (0.1 mg/mL) to kitten-formula (mixed three to one with cow milk) to six bottle-feed kittens for 5 days. This study reported that kittens fed with kitten-formula supplemented with hBSSL had a growth rate of twice that of those fed with formula alone. Of note is that the formula was supplemented with cow milk, the kittens were not preterm or of low birth weight, they were breast fed for the first 48 hours of their life and the study was conducted with purified native hBSSL. The authors suggested that the kitten could be utilized as an animal model in the investigation of the functional role of BSSL, and on the basis of this study related patent applications were filed (including, U.S. Pat. No. 4,944,944, EP 0317355 and EP 0605913) that disclose (amongst other aspects): a method for fortifying a fat-containing infant formula which is poor in bile-salt-activated lipase comprising adding to the formula an effective amount of an isolated bile-salt-activated lipase selected from the group consisting of milk bile-salt-activated lipase [BSSL] and bile-salt-activated pancreatic carboxylesterase [now known also to be BSSL] to increase fat absorption from the formula and growth of the infant; and a method for feeding an infant a dietary base from a first source comprising fats consisting of administering an isolated bile-salt-activated lipase selected from the group consisting of milk bile-salt-activated lipase [BSSL] and bile-salt-activated pancreatic carboxylesterase [also BSSL] to the infant in an amount sufficient to improve the infant's digestion and absorption of the fats in the base and increase the growth of the infant, wherein the lipase is derived from a second source. No data supporting an improvement in fat absorption were disclosed, nor any data obtained from any study that involved human infants. Another study (Lindquist et al, 2007; J Pediatr Gastroenterol Nutr 44: E335) has been reported by Lindquist & Hernell (2010) as artificially feeding purified human BSSL to BSSL-knock-out mice pups nursed by BSSL-knock-out dams to restore normal fat absorption and preventing the formation of intestinal lesions.
Following the cloning of the hBSSL cDNA and the disclosure of various approaches to produce large quantities of recombinant human BSSL (rhBSSL), numerous disclosures have been made, and claims to, various infant formulas comprising rhBSSL (for example, U.S. Pat. No. 5,200,183, WO 91/15234, WO 91/18923, and U.S. Pat. No. 5,716,817) and various methods or uses of such formula or rhBSSL, including as an infant supplement, for the improvement of utilization of dietary lipids, treatment of fat malabsorption, certain pancreatic abnormalities and cystic fibrosis (for example, WO 91/18923, WO 94/20610 and WO 99/54443). However, as with the earlier suggestive studies, no supporting data obtained from experiments supplementing human infants with recombinant bile-salt-stimulated lipase are disclosed. Indeed, in 1996 after all these suggestions, associative studies and disclosures, leading workers in the area were still questioning: “Should bioactive components of human milk [such as BSSL] be supplemented to formula-fed infants?”; and further stating that: “There are no data on attempts to supplement digestive enzymes [such as BSSL]” (Hamosh, at Symposium: Bioactive Components in Milk and Development of the Neonate: Does Their Absence Make a Difference? Reported in J Nutr, 12: 971-974; 1997). More recently, Andersson & coworkers (2007) have speculated that supplementing pasteurized milk with recombinant human milk BSSL may restore endogenous lipolytic activity of the milk.
The 722 amino-acid native BSSL is heavily glycosylated (30-40% carbohydrate) (Abouakil et al, 1989; Biochem Biophys Acta, 1002: 225-230), with extensive O-glycosylation sites within the C-terminal portion of the molecule that in its most abundant form contains 16 proline-rich repeats of 11 residues with O-linked carbohydrates (Hansson et al, 1993; J Biol Chem, 268: 26692-26698). The role of the extensive O-glycosylation is unproven, but based on its sequence composition the large C-terminal tail is predicted to be very hydrophilic and accessible (Wang et al, 1995; Biochemistry, 34: 10639-10644).
Differences in glycosylation patterns can have dramatic differences in the activity or other properties of many proteins, especially proteins used in medicine. For example, ARANESP (darbepoetin alpha) is a specifically engineered variant of erythropoietin that differs from PROCRIT (epoetin alpha) by 2 amino acids that provides the molecule with 5 N-linked oligosaccharide chains rather than 3, and which significantly alter the pharmacokinetic properties; with darbepoetin showing a threefold increase in serum half-life and increased in vivo activity compared to epoetin (Sinclair and Elliot, 2005; J Pharm Sci 94: 1626-1635).
Different recombinant production systems (such as mammalian cell, yeast, transgenic animal), and even seemingly minor changes in production process from the same expression system, can lead to changes in the glycosylation of the same protein/polypeptide sequence. For example, recombinant human alpha-galactosidase A is used in enzyme replacement therapy for Fabry's disease, and the commercial drug product is produced in two ways, having the same amino acid sequence but each having a different glycosylation pattern: REPLAGAL (agalsidase alfa) and FABRAZYME (agalsidase beta). REPLAGAL is produced in a continuous line of human fibroblasts while FABRAZYME produced in Chinese hamster ovary (CHO) cells, and each product has different glycosylation. In common with other proteins produced from CHO cells, FABRAZYME is a sialyated glycoprotein, and has differences in the degree of sialyation and phosphorylation compared to REPLAGAL (Lee et al, 2003; Glycobiology, 13: 305-313). The qualitative and quantitative differences in the sialylation of glycoproteins produced in CHO cells in comparison with natural human glycoproteins have consequences for both the level of biodistribution and immunogenic potency. In fact, the presence of IgG has been reported in almost all patients treated with agalsidase beta compared to only 55% of patients treated with agalsidase alfa (Linthorst et al, 2004; Kidney Int, 66: 1589-1595). Moreover, in some cases, an allergic type reaction to treatment with agalsidase beta has been recorded, with the presence of IgE in the circulation and/or a positive intradermal reaction (Wilcox et al, 2004; Am J Hum Genet, 75: 65-74).
Indeed, while their peptide maps are very similar, the glycosylation patterns of native BSSL does differ substantially from that of rhBSSL produced in mouse C127 and hamster CHO cell lines, and also in the ability to bind to certain lectins including concanavalin, Ricinus communis agglutinin and Aleuria aurantia agglutinin suggesting that native BSSL contains considerably more fucose and terminal beta-galactose residues than the recombinant forms (Stromqvist et al, 1995; J Chromatogr, 718: 53-58). Landberg et al (1997; Arch Biochem Biophys 344: 94-102) further characterized these two recombinant forms, and reported that both recombinant forms had a lower molar percent of total monosaccharide (20% and 15% for C127- and CHO-produced rhBSSL, respectively, compared to 23% for native hBSSL), and that while native hBSSL reacted to certain Lewis antigen-detecting antibodies, the C127-rhBSSL did not.
Although the C127- and CHO-produced rhBSSL reported above were generally similar to each other in terms of molecular mass, glycosylation and lectin binding, in contrast, the rhBSSL isolated from the milk of transgenic mice showed a lower apparent molecular mass on size-exclusion chromatography (SEC) and no detectable interactions with a panel of lectins, indicating a significantly lower degree of O-glycosylation of rhBSSL in milk from transgenic mice than found for the other recombinant forms (Stromqvist et al, 1996; Transgen Res 5: 475-485).
Clinical studies in specific indications conducted with one particular form of rhBSSL have been reported; namely early-phase exploratory studies of exocrine pancreatic insufficiency (PI) due to chronic pancreatitis or cystic fibrosis (CF). In 2004, a phase II trial was reported that showed that CF patients (aged 12 to 39 years) with PI had a more rapid and efficient lipid uptake when supplemented with rhBSSL at a single dosing of 0.2 g or 1 g as a complement to 25% of their regular Creon dosing, as compared to Creon alone given at their regular dose, or at 25% dosage (Strandvik et al, 2004; 18th North American Cystic Fibrosis Conference, St Louis Mich.; abstract published in Pediatr Pulmonol, S27: 333), and in 2005 the results of a second phase II trial were reported as rhBBSL showing a greatly improved ability of a group of Swedish patients with CF suffering from PI to digest fat (press release from Biovitrum, reporting Strandvik et al, 2005; 28th European Cystic Fibrosis Society (ECFS) Conference, Crete). In both clinical trials, these clinical results were obtained using rhBSSL-OVI. More recently, it has been announced that a further phase II trial with an oral suspension of rhBSSL (described therein as “bucelipase alpha”), dosed at 170 mg 3 times daily for 5-6 days, to evaluate the effect on the fat absorption in adult patients with CF and PI has been completed, but no efficacy results from this have to date been published (clinicaltrials.gov identifier NCT00743483).
It has been disclosed since at least 2008 that two phase II trials using rhBSSL were planned and ongoing, each to investigate the coefficient of fat absorption, and change in length and body weight, in preterm infants born before 32 weeks gestational age treated with 0.15 g/L rhBSSL or placebo for one week each, added to infant formula (clinicaltrials.gov identifier NCT00658905) or to pasteurized breast milk (clinicaltrials.gov identifier NCT00659243).
In light of the prior art, and the long felt need for a solution, it is therefore an object of the present invention to provide a method of increasing the growth velocity of a human infant, such as an underweight or preterm human infant. Said method should overcome one or more of the disadvantages of the prior art, that include: that an active ingredient that can be reliably and/or reproducibly produced in large quantities; that the active ingredient has been manufactured by a scientifically, regulatory and/or ethically acceptable method; and/or that the method or the active ingredient used in the method, has been demonstrated within a randomized clinical trial involving human infants to be efficacious and safe.
The solution to the above technical problem is provided by the various aspects and embodiments of the present invention as defined or otherwise disclosed herein and/or in the claims.